Effective cancer immunotherapy appears to be dependent on creating the right inflammatory reaction at the site of the tumor. Preliminary and published data provide compelling evidence that experimentally creating a hyperthermic state, similar to that achieved during a common natural event (fever) may have the potential to augment tumor control by the immune system. The overall hypothesis of this proposal is that thermal (fever-range) stimulation of the inflammatory activity of granulocytes, or polymorphonuclear neutrophils (PMNs), helps to eliminate cancer cells in the body and is at least part of the basis by which a mild, fever-range hyperthermia helps to control tumor growth.. The specific aims of this grant proposal have been designed to obtain cellular and molecular data that will test this hypothesis and help to identity potential mechanisms by which the thermal microenvironment enhances PMN activity. Aim 1 will utilize both syngeneic tumor models and human tumor xenograft models in mice to quantify the kinetics of hyperthermia-induced PMN localization to the tumor site. The specific contribution of PMNs to the anti-tumor effects of fever-range whole body hyperthermia (FR-WBH) will also be studied using antibody-blocking experiments and mice with PMNs that are defective in their homing function. Using various in vitro functional assays and flow cytometric analysis of activation and maturation markers, Aim 2 will then more directly test the hypothesis that FR-WBH exerts its anti-tumor effect through PMNs by increasing their migratory potential and/or increasing the overall activation status of these cells. Lastly, Aim 3 will determine whether thermally-treated PMNs exert tumoricidal activity through the production of reactive nitrogen or oxygen intermediates. Overall, this pilot research program should help to pinpoint an important role for PMNs in the effector activity of FR-WBH. Specifically, these studies will provide sufficient preliminary data for a future RO1 application that will examine even more closely the molecular mechanisms and/or signaling pathways by which fever-range temperatures regulate the PMN activities as defined in this R21 application. By obtaining a better understanding of the molecular mechanisms by which the thermal microenvironment regulates inflammatory responses, a more rational approach can be made in the use and design of future clinical trials that will utilize hyperthermia as a complimentary or alternative cancer therapy. Moreover, studies such as this can provide well-characterized cellular endpoints by which the responsiveness of individual patients to thermal therapy can be objectively measured and compared, an essential practice for validating any nature-based immunotherapy such as FR-WBH in which the responses of individuals may vary.